Directed evolution of a far-red fluorescent rhodopsin

Abstract

Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with livecell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK a of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼620 nm/730 nm).optogenetics | opsins | bioelectricity | voltage sensor | near-infrared

Cellular Level Brain Imaging in Behaving Mammals: An Engineering Approach

“…However, in brain slices Arch’s dim fluorescence necessitates intense illumination (~12 W/mm 2 ) that excites tissue auto-fluorescence and heats the specimen (Hochbaum et al, 2014). Toward enabling studies in behaving mammals, efforts to create brighter Arch variants are underway (Flytzanis et al, 2014; Hochbaum et al, 2014; McIsaac et al, 2014). …”

Methods for the directed evolution of proteins

“…Although these strategies can be applied to multigene pathways 3,4 and gene networks [5][6][7] , the examples in this Review will focus exclusively on the laboratory evolution of single genes. In addition, although many of these approaches apply to other types of biomolecules, we focus on the directed evolution of proteins because protein evolution has proved to be especially useful for generating novel biocatalysts 8 , reagents 9 and therapeutics 10 .…”

Microcircuits in Epilepsy: Heterogeneity and Hub Cells in Network Synchronization

“…The development of miniaturized microscopes that can be implanted to observe cell dynamics during freely mov-ing behavior (Wilt et al 2009;Ghosh et al 2011;Ziv et al 2013) has provided opportunities to study microcircuit alterations in chronically epileptic animals. New optogenetic tools are also expanding at an astounding pace-opsins that are more sensitive or have different channel dynamics are allowing for even finer manipulation of network activity (Chuong et al 2014;Dhakal et al 2014;Hochbaum et al 2014;McIsaac et al 2014). As experimental methods progress, computational tools for modeling neural networks likewise continue to become more complex and powerful.…”